Novel use of regulatory t cell-specific surface protein lrig-1

ABSTRACT

The present invention relates to a novel use of regulatory T cell-specific surface protein Lrig-1, and more specifically to an immunosuppressive agent comprising siRNA which inhibits the expression of surface protein Lrig-1. In addition, the invention relates to a method for screening an immunosuppressive agent which inhibits proteins of Lrig-1 or genes encoding the proteins. As a result, an immunosuppressive agent with low side effects and high specificity can be developed.

PRIORITY

This application is a continuation application of U.S. Ser. No.14/427,421, filed on Mar. 11, 2015, which is national phase which is a371 National Stage Application that claims priority to InternationalPatent Application No. PCT/KR2012/00532, filed on Jan. 20, 2012, whichclaims the benefit of KR10-2011-0007022, filed on Jan. 24, 2011, all ofwhich are incorporated in their entirety by reference herein.

TECHNICAL FIELD

The present invention relates to a novel use of regulatory Tcell-specific surface protein Lrig-1.

BACKGROUND ART

The inflammatory and immune responses are a very important biologicalphenomenon that protects the body from outside harmful substances(bacteria, virus) using various inflammatory cells and immune cells,among which the T cells play an important role like the brain that makesan overall regulation for the inflammatory and immune responses. It hasbeen known that the human with a variety of different genetic andenvironmental factors may naturally produce excessive inflammatory andimmune responses and that the internal risk factors (high cholesterol,blood sugar, etc.) threatening the health and safety of a living bodymay cause chronic inflammation and immune hypersensitivity reactions,which lead to autoimmune diseases (asthma, atopy, rheumatoid arthritis,Crohn's disease, MS) and chronic inflammatory diseases (myocardialinfarction, diabetes, degenerative cerebrovascular diseases), finallyleading to the death.

The regulatory T cell (Treg), one subset in T cells of the body, playsan important role to naturally prevent the occurrence of excessiveinflammation and immune responses, but if autoimmune diseases andchronic inflammatory diseases are occurred, the function and number ofthe regulatory T cells have been known to be rather significantlyreduced. Therefore, for patients with immune diseases and inflammatorydiseases, generating a lot of the regulatory T cells is the mostimportant and natural therapy. Further, in the recent studies, cancercells have been known to inhibit the natural immunity of cancer cells ina living body by increasing the number and function of the regulatoryT-cells (Treg) around them, thus allowing the occurrence, progressionand metastasis of cancers.

Until now, a few regulatory T cell (Treg)-specific genes and proteinshave been developed and published.

In studies to date, the Treg has been known to express a high level ofCD25. CD25 constitutes heterotrimer IL-2 receptor with α,β,γ-chains.This has been known to have more than 100 fold affinity to IL-2 ascompared to β, γ-chain heterodimer IL-2 receptors existed inconventional non-contacted T cells. Through competition for IL-2 greatlyinvolved in T cell proliferation, the proliferation of non-contacted Tcells is inhibited to control the immune response. IL-2 has been used aslabeled materials for the regulatory T cells up to now, but IL-2receptor with a high level of homologous relation is expressed in a highlevel upon activation of the different types of T cells, and thus it isdifficult to become definite labeling materials.

It has been known that since Treg express CTLA4, they combine with CD80or CD86 expressed in the other immune T cells or antigen-presentingcells to thereby inhibit the activation thereof. However, recently, whenthe different types of immune T cells such as T helper 1 or T helper 2cells are also activated and, after passing a certain time, theexpression of IL-2 is reduced, it has been reported that the expressionof CTLA4 is increased. It has been also known that CTLA4 are also notthe material specifically expressed in Treg.

Therefore, in order to differentiate the regulatory T cells, alternativelabeling materials such as low expression levels of CD62L, CD38, CD103,GITR and CD45RB have been proposed. As it has been discovered that,among them, the transcription factor, Foxp3 (Forkhead box 3), plays adecisive role in the differentiation and activity of natural CD4+regulatory T cells, this has been known to be the most effectivelabeling material. However, as the expression of Foxp3 in CD4+IL-10+Tr1cells which exhibit immunoregulatory functions in the peripheral hasbeen found to be not essential, the potential as the regulatory T cell(Treg)-specific markers of Foxp3 has been gradually diluted (see,IL-35-mediated induction of a potent regulatory T cell population, LarenW Collison et al., Vol. 11, Nature Immunology, 1093-1952, 2010).Furthermore, the technology of controlling the function or expression ofFoxp3 to thereby control the function of the regulatory T cells (Treg)has not been known up to now. Because of molecular biologicalcharacteristics that Foxp3 is a transcription factor, it is not possibleto use Foxp3 as a marker for the development of medicines for treatingdiseases and the separation of Treg cells using Foxp3.

On the other hand, LRIG1 (leucine-rich and immunoglobulin-likedomains 1) protein is a transmembrane protein, and LRIG1 gene is highlyexpressed in normal skin and expressed in basal and follicular cells toregulate the proliferation of epithelial stem cells. Therefore, the LRIGis important in maintaining the homeostasis of the epidermis and theabsence thereof may develop psoriasis or skin cancer. The gene family ofLRIG is present in three types, i.e., LRIG1, LRIG2 and LRIG3. The aminoacid sequence homology between these families is more than 60% (forLRIG1 and LRIG2) and more than 40% (for LRIG1 and LRIG3). The amino acidsequence of important functional domain maintains substantially 100%. Assuch, LRIG2 and LRIG3 are also anticipated to have a lot of functionalsimilarity. Further, as shown in Example 10 of the present invention,LRIG1 is specifically highly expressed even in the regulatory T cells ofthe human. In particular, the expression has been shown to increase inthe activated or proliferated regulatory T cells. It has been reportedthat cutting off the chromosome 3p14.3 part at which LRIG1 is positionedis highly likely to develop into cancer cells. Actually, it has beenidentified that the expression of LRIG1 is greatly reduced in renal cellcarcinoma and cutaneous squamous cell carcinoma. Currently, LRIG1induces the ubiquitination of EGFR (Epidermal Growth Factor Receptor)via c-Cbl to degrade a protein and so blocks the signal transduction byphosphorylation of MAPK and AKT which is present in the lower part andinvolved in cell proliferation and increases the secretion of caspase-8,thus leading to apoptosis. In this regard, the potential as a cancerinhibitor has been suggested.

The present inventor has conducted numerous researches and experimentsin order to find out genes or proteins specifically expressed in theregulatory T cells, and discovered that Lrig-1 is specifically expressedin the regulatory T cells through bioinformatics approaches calledmicroarray, proteomics and functional gene network and that theexpression is important in the function, differentiation and growth ofthe regulatory T cells (Treg). The present invention has been completedon the basis of such discovery.

DISCLOSURE Technical Problem

The present invention provides a method for screening immunosuppressiveagents or immune activators which comprises the steps of (a) contactingan analyzing sample to a regulatory T cell including Lrig (leucine-richrepeats and immunoglobulin-like domains) protein; (b) measuring theamount or activity of the protein; and (c) determining that the sampleis the immunosuppressive agents or the immune activators when the amountor activity of the protein is measured to be down- or up-regulated.

The present invention also provides a composition for treatingimmune-related diseases comprising Lrig 1 protein.

Technical Solution

In one embodiment of the present invention, there is provided a methodfor screening immunosuppressive agents or immune activators whichcomprises the steps of (a) contacting an analyzing sample to aregulatory T cell including Lrig (leucine-rich repeats andimmunoglobulin-like domains) protein; (b) measuring the amount oractivity of the protein; and (c) determining that the sample is theimmunosuppressive agents or the immune activators when the amount oractivity of the protein is measured to be down- or up-regulated. TheLrig protein may be any one selected from the group consisting of Lrig1, Lrig 2 or Lrig 3. Further, the Lrig protein is set forth in SEQ IDNO: 11.

In one embodiment of the present invention, there is provided a methodfor screening immunosuppressive agents or immune activators whichcomprises the steps of (a) contacting an analyzing sample to aregulatory T cell (Treg) including LRIG gene; (b) measuring theexpression level of the gene; and (c) determining that the sample is theimmunosuppressive agents or the immune activators when the expressionlevel of the gene is measured to be down- or up-regulated. The LRIG genemay be a nucleotide encoding SEQ ID NO: 11. Further, the LRIG gene maybe a nucleotide as set forth in SEQ ID NO: 12.

In another embodiment of the present invention, there is provided amethod for screening immunosuppressive agents or immune activators forcombination administration which comprises (a) the first step ofcontacting the immunosuppressive agents or the immune activators tocells including Lrig 1 protein as set forth in SEQ ID NO: 11 and thenmeasuring the amount or activity of the protein; (b) the second step ofcontacting an analyzing sample, the immunosuppressive agents or theimmune activators to cells including Lrig 1 protein as set forth in SEQID NO: 11 and then measuring the amount or activity of the protein; and(c) the step of comparing the measurement values of the first and secondsteps and then determining that the sample is the immunosuppressiveagents or the immune activators for combination administration when themeasurement value of the second step is down- or up-regulated ascompared to the measurement value of the first step. Theimmunosuppressive agents may be any one selected from the groupconsisting of Glucocorticoids, Cyclophosphamide, Cyclosporin,Tacrolimus, Rapamycin, Type IV PDE inhibitors, p38 kinase inhibitors,Azathioprine, mycophenolate mofetil, Mizoribin, Methotrexate,Leflunomid, Brequina and Methotrexate.

In one embodiment of the present invention, there is provided a methodfor screening immunosuppressive agents or immune activators forcombination administration which comprises (a) the first step ofcontacting the immunosuppressive agents or the immune activators tocells including LRIG1 gene as set forth in SEQ ID NO: 12 and thenmeasuring the expression level of the gene; (b) the second step ofcontacting an analyzing sample, the immunosuppressive agents or theimmune activators to cells including LRIG1 gene as set forth in SEQ IDNO: 12 and measuring the expression level of the gene; and (c) comparingthe measurement values of the first and second steps and determiningthat the sample is the immunosuppressive agents or the immune activatorsfor combination administration when the measurement value of the secondstep is down- or up-regulated as compared to the measurement value ofthe first step. The immunosuppressive agent may be any one selected fromthe group consisting of Glucocorticoids, Cyclophosphamide, Cyclosporin,Tacrolimus, Rapamycin, Type IV PDE inhibitors, p38 kinase inhibitors,Azathioprine, mycophenolate mofetil, Mizoribin, Methotrexate,Leflunomid, Brequina and Methotrexate.

In one embodiment of the present invention, there is provided animmunosuppressive agent comprising, as an active ingredient, anantisense having a sequence complementary to the nucleotide sequence asset forth in SEQ ID NO: 12 or siRNA (small interference RNA)oligonucleotide. The siRNA may be any one of siRNA as set forth in SEQID NO: 7, 8, 9, or 10. The immunosuppressive agents are characterized bytreating any one disease selected from the groups consisting ofautoimmune diseases, graft versus host diseases, organ transplantrejection, asthma, atopy, acute and chronic inflammatory diseases,cardiovascular diseases and cognitive disorders.

In one embodiment of the present invention, there is provided a kit fordiagnosis of immune-related diseases comprising all or part of theantibody specifically binding to Lrig1 protein. The Lrig1 protein may beset forth in SEQ ID NO: 11. Also, the immune-related diseases may be anyone selected from the group consisting of an autoimmune disease, graftversus host diseases, organ transplant rejection, asthma, atopy, acuteand chronic inflammatory diseases, cardiovascular diseases and cognitivedisorders.

In one embodiment of the present invention, there is provided a kit fordiagnosis of immune-related diseases comprising all or part of theantibody specifically binding to a cell membrane surface protein ofLrig1 protein. The cell membrane surface protein of Lrig1 protein may beset forth in SEQ ID NO: 13. Also, the immune-related diseases may be anyone selected from the group consisting of autoimmune diseases, graftversus host diseases, organ transplant rejection, asthma, atopy, acuteand chronic inflammatory diseases, cardiovascular diseases and cognitivedisorders.

In one embodiment of the present invention, there is provided acomposition for treating immune-related diseases comprising Lrig1protein. The Lrig1 protein may be set forth in SEQ ID NO: 11. Also, theimmune-related diseases may be any one selected from the groupconsisting of autoimmune diseases, graft versus host diseases, organtransplant rejection, asthma, atopy, acute and chronic inflammatorydiseases, cardiovascular diseases and cognitive disorders.

In one embodiment of the present invention, there is provided acomposition for treating immune-related diseases comprising a cellmembrane surface protein of Lrig1 protein or a protein including all orpart of the antibody specifically binding thereto. The cell membranesurface protein of Lrig1 protein may be set forth in SEQ ID NO: 13.Also, the immune-related disease may be any one selected from the groupconsisting of autoimmune diseases, graft versus host diseases, organtransplant rejection, asthma, atopic dermatitis, acute and chronicinflammatory diseases, cardiovascular diseases and cognitive disorders.

In one embodiment of the present invention, there is provided acomposition for anti-cancer treatment comprising Lrig1 protein. TheLrig1 protein may be set forth in SEQ ID NO: 11.

In one embodiment of the present invention, there is provided acomposition for anti-cancer treatment comprising a cell membrane surfaceprotein of Lrig1 protein or a protein including all or part of antibodyspecifically binding thereto. The cell membrane surface protein of Lrig1protein may be set forth in SEQ ID NO: 13.

In one embodiment of the present invention, there is provided acomposition for promoting vaccination comprising Lrig1 protein. TheLrig1 protein may be set forth in SEQ ID NO: 11.

In one embodiment of the present invention, there is provided acomposition for promoting vaccination comprising a protein including acell membrane surface protein of Lrig1 protein or all or part ofantibody specifically binding thereto. The cell membrane surface proteinof the Lrig1 protein may be set forth in SEQ ID NO: 13.

In one embodiment of the present invention, there is provided a methodfor screening regulatory T cells overexpressing Lrig1 protein; and amethod for treating immune-related diseases comprising administering aneffective amount of the screened regulatory T cells to a subject withimmune-related diseases. The immune-related diseases may be any oneselected from the group consisting of autoimmune diseases, graft versushost diseases, organ transplant rejection, asthma, atopic dermatitis,acute and chronic inflammatory diseases, cardiovascular diseases andcognitive disorders.

In one embodiment of the present invention, there is provided a methodfor treating an immune-related disease comprising administering aneffective amount of a Lrig1 protein, or a protein comprising a cellmembrane surface protein of the Lrig1 protein, or a compositioncomprising all or part of the antibody specifically binding thereto to asubject with the immune-related diseases. The Lrig1 protein may be setforth in SEQ ID NO: 11. The cell membrane surface protein of Lrig1protein may be set forth in SEQ ID NO: 13. The immune-related diseasesmay be any one selected from the group consisting of autoimmunediseases, graft versus host diseases, organ transplant rejection,asthma, atopic dermatitis, acute and chronic inflammatory diseases,cardiovascular diseases and cognitive disorders.

In one embodiment of the present invention, there is provided a methodfor screening regulatory T cells overexpressing Lrig1 protein; and amethod for treating caners comprising administering an effective amountof the screened regulatory T cells to a subject with the cancers.

In one embodiment of the present invention, there is provided a methodfor treating cancers comprising administering an effective amount of aLrig1 protein, or a protein comprising a cell membrane surface proteinof Lrig1 protein, or a composition including all or part of the antibodyspecifically binding thereto to a subject with the cancers. The Lrig1protein may be set forth in SEQ ID NO: 11. The cell membrane surfaceprotein of the Lrig1 protein may be set forth in SEQ ID NO: 13.

In one embodiment of the present invention, there is provided acomposition for promoting vaccination comprising administering a Lrig1protein, or a protein comprising a cell membrane surface protein of theLrig1 protein to a subject. The Lrig1 protein may be set forth in SEQ IDNO: 11. The cell membrane surface protein of Lrig1 protein may be setforth in SEQ ID NO: 13.

The term “immune-related disease” as used herein refers to diseasesinduced by excessive activation or inhibition of various immune cellsand inflammatory cells, for example, but is not limited to, autoimmunediseases, graft versus host diseases, transplant rejection, asthma,atopy, acute and chronic inflammatory diseases, cardiovascular diseaseand cognitive disorders.

In the present invention, Lrig1 (leucine-rich and immunoglobulin-likedomains 1)″ is a transmembrane protein consisting of 1091 amino acids,and consists of leucine repeat sequence (leucine-rich repeat (LRR)) ofthe extracellular or lumen side and three immune antibody-like domain(immunoglobulin-like domains), cell transmembrane sequences andcytoplasmic tail part. The LRIG gene family consists of LRIG1, LRIG2 andLRIG3, and amino acids between them are very conservative.

In the present invention, “siRNA (small interfering RNA)” are a class ofdouble-stranded RNA duplex with short 19-30 ribonucleic acid chains,which exhibits the effects of inhibiting only the expression of acertain gene without non-specific inhibition if introduced within cells.The mechanism of action of siRNA has been known that siRNA combines withRISC (RNA-induced silencing complex) within cells, the sense strand ofgenes corresponding to mRNA is cut off and the antisense standcomplementary to the sense strand is present as a complex with RISC andthen combined with mRNA having the complementary base sequence, that is,mRNA of the target gene to decompose them, thereby inhibiting theexpression of the gene.

In the present invention, “shRNA (short hairpin RNA)” means that oligoDNA connecting 3-10 base linkers are synthesized between the sense andcomplementary nonsense of target gene siRNA sequences and then cloned toplasmid vector, or shRNA is inserted and expressed into retrovirus suchas lentivirus and adenovirus to thereby make shRNA of hairpin structurewith loop, which is converted into siRNA via Dicer within cells, thusexhibiting RNAi effects. shRNA has advantages that it has RNAi effectsfor a relatively long-term. siRNA of the present invention may be achemically modified form to prevent the rapid degradation by in vivonuclease. Since siRNA has a double helix structure, which is arelatively stable structure as compared with ribonucleic acids orantisense oligonucleotides having a single helix structure, but it israpidly degraded by the in vivo nuclease. Therefore, the siRNA canreduce the degradation speed through a chemical modification. The methodfor chemically modifying siRNA into stable and resistant forms so thatsiRNA is not easily degraded have been well known to a person skilled inthe art.

The most common method used for the chemical modification of siRNA isthe method for modification of boranophosphate or phosphorothioate.These materials stably form a connection between the nucleosides ofsiRNA, thus imparting a resistance to nucleic acid decomposition. Theribonucleic acid modified with boranophosphate has characteristic thatthe degradation of the nucleic acid is not well made. However, thisribonucleic acid is not made by a chemical reaction, but synthesizedonly by the manner wherein boranophosphate enters into the ribonucleicacid by in vitro transcription reaction.

The method for modification of boranophosphate is relatively easy, buthas a drawback which is difficult to modify at a particular position. Onthe other hand, the method for modification of phosphorothioate has anadvantage that can introduce a sulfur atom in a desired portion.However, a severe degree of phosphothioation may appear problems, forexample, decrease of efficacy, toxicity, non-specific RISC (RNAinducedsilencing complex) formation, etc.

Therefore, recently, in addition to the above two methods, the method ofconducting the chemical modification only at the end position (the 3′terminal exceeding part) to impart resistance to the nuclease is morepreferred. It is also known that, although ribose ring is chemicallymodified, resistance to the nuclease become strong. In particular,variations of the ribose 2′-position in the original cells willstabilize siRNA. However, when the methyl group exactly enters thisposition, the stability is increased, whereas too many methyl groups maycause problems, for example, loss of RNA-mediated interference. Thepurpose of such chemical modification is to increase the efficacy andthe pharmacokinetic residence time in vivo (see Mark et al., MolecularTherapy, 13: 644-670, 2006). In addition to the chemical modification,in order to increase the delivery efficiency within cells of siRNA, asafe and efficient delivery system is required. To this end, siRNA ofthe present invention may be included in the pharmaceutical compositionfor the treatment of cancers in a complex form with nucleic aciddelivery system.

The nucleic acid delivery system for delivering a nucleic acid substanceinto cells may be greatly divided into a viral vector and a non-viralvector. The most widely used system is a viral vector because thedelivery efficiency is high and the time of duration is long. Amongvarious viral vectors, retroviral vector, adenoviral vector,adeno-associated viral vector and the like are mainly used. These viralvectors are efficient in view of the cell delivery of the ribonucleicacid, but it has a number of problems in view of safety such as arecombinant into virus having activity in a living body, induction ofthe immune response, random insert into host chromosome, and the like.In contrast, nonviral vectors have the advantages over the viralvectors, that is, the immune response to the toxicity is low, repeatedadministration is possible, formation of a complex with RNA is simple,and mass production is easy. Also, a specific ligand at a disease cellor a tissue site is conjugated to a non-viral vector, thus allowing fora long-term cell selective nucleic acid delivery. As the non-viralvectors, a variety of formulations, such as micelles, emulsions,nanoparticles, including liposomes and cationic polymers, can be used.The nucleic acid delivery system can significantly enhance the transportefficiency of the desired nucleic acid in animal cells and the nucleicacid delivery can be made even to any animal cell in accordance with theintended use of the nucleic acid to be delivered.

The monoclonal antibodies to the protein of the present invention may beproduced and used by a conventional method for producing monoclonalantibodies in the technical field to which the present inventionpertains. Typically, the commercially available antibodies can be used.Monoclonal antibodies to the proteins can be quantitatively analyzed byconducting a color reaction using the secondary antibodies andsubstrates thereof conjugated with enzymes such as alkaline phosphatase(AP) or horseradish peroxidase (HRP). Alternatively, it can bequantitatively analyzed using those where AP or HRP enzymes areconjugated directly to the monoclonal antibodies to the proteins. Inaddition, the polyclonal antibodies which recognize the protein in placeof the monoclonal antibodies can be used. The polyclonal antibodies canbe produced and used by a conventional method for producing antiserum inthe technical field to which the present invention pertains.

The protein of the present invention comprises a fusion protein, whichis expressed by combining or linking at least two different proteinsthrough a method of genetic engineering, and which has two or morefunctions. In particular, in the case of an antibody, themulti-functional antibody can be produced by replacing Fab region or Fcregion with other protein.

In the screening method of the present invention, the reaction betweenthe composition comprising the protein and the testing substances can beidentified using a conventional method which is used to identify thereaction between protein-protein and protein-compound. For example, themethod of measuring the activity after the reaction of the protein andthe testing substance, yeast two-hybrid and the like can be used.

Search of phage-displayed peptide clone binding to the protein, highthroughput screening (HTS) using natural products and chemicallibraries, drug hit HTS, cell-based screening or screening method usingDNA array can be used. In this case, the composition of the presentinvention may comprise, in addition to the protein, a buffer or reactionsolution to stably maintain a structure or physiological activity of theprotein. In addition, the compositions of the present invention maycomprise, for the in vivo experiments, cells expressing the protein, orcells containing a plasmid expressing the protein under a promotercapable of controlling the transcription ratio.

In the screening method of the present invention, the testing substancesused here may include individual nucleic acids, proteins, other extractsor natural products, compounds, etc., which are assumed to have apotential as a cancer metastasis inhibitor according to the conventionalscreening manner or randomly selected. Further, it may include thetesting substance which is obtained through the screening method of thepresent invention and which exhibits activities to promote the geneexpression or enhance the function of the protein, and inversely thetesting substance showing an activity to inhibit the expression of thegene or inhibit the function of the protein. The former may be acandidate immunosuppressive agent by developing an inhibitor into thetesting substance, and the latter may be a candidate immunosuppressiveagent. Such candidate immunosuppressive agent acts as a leading compoundin the subsequent development process of the immunosuppressive agent. Anew immunosuppressive agent can be developed by transforming andoptimizing the structure so that the leading compound can exhibit thesuppressive effect of the gene or protein expressed therefrom.

The immune related diseases can be treated using the protein or theantibody of the present invention. That is, in the case of autoimmunediseases, when ERK is activated with the selectively produced ERKactivator, the resistant dendritic cells are activated, therebyproliferating the regulatory T cell and increasing the number of theproliferation, the result of which confirms the effect of RA treatment(see Arthritis Rheum. 2011 January; 63 (1):84-95, “selective ERKactivation differentiates mouse and human tolerogenic dendritic cells,expands antigen-specific regulatory T cells, and suppresses experimentalinflammatory arthritis”). When treated to mice which induced RA byinjection of type II collagen using Anti-CD3, the numbers ofCD4+CD25+Foxp3+Regulatory T cells and CD8+CD25+Foxp3+Regulatory T cellswere greatly increased, and so it can be used in the treatment of RA(see Arthritis Rheum. 2010 January; 62 (1): 171-8, “ANTI-CD3 therapyexpands the numbers of CD4+ and CD8+Treg cells and induces sustainedamelioration of collagen-induced arthritis”). When TCR BV8S2 CDR2peptide found to do an immunosuppressive action was treated to thenon-transgenic C57BL/6 EAE model, EAE was greatly suppressed. This isbecause the immune action was controlled as the number of regulatory Tcells rapidly increased, thereby improving the symptom (see Immunology.2012 February; 135 (2):168-79, “Regulatory T cells play a role in T-cellreceptor CDR2 peptide regulation of experimental autoimmuneencephalomyelitis”). In the case of patients suffering from IBD, theregulatory T cells become self-apoptosis to cause damage to the immuneregulatory system. Therefore, it has been observed that in order tosuppress this, the apoptosis of regulatory T cells is increased inchronic inflammatory bowel diseases by anti-TNF-alpha treatment (seeGut. 2011 October; 60 (10):1345-53. Epub 2011 Apr. 1, “Apoptosis ofregulatory T lymphocytes is increased in chronic inflammatory boweldisease and reversed by anti-TNFα treatment”). When12-O-tetradecanoylphorbol 13-acetate to induce psoriasis was treated tothe mice prepared by conducing knockout (Pglyrp2), it was confirmed toinduce the more severe inflammation response than the wild type mice.

The mechanism that Pglyrp2 controls the inflammatory response wasconfirmed to limit Th17 responses and, at the same time, promote andactivate the regulatory T cells (see J Immunol. 2011 Dec. 1; 187(11):5813-23. Epub 2011 Nov. 2, “Peptidoglycan recognition proteinPglyrp2 See protects mice from psoriasis-like skin inflammation bypromoting regulatory T cells and limiting Th17 responses”).

On the other hand, it has been found that the treatment was possible byreducing the number of CD4+ T cells in colorectal cancer (CRC), but thetreatment was not made. The reason is that the regulatory T cells wereover-expressed, thereby suppressing the immune function to remove thetumor cells. In order to confirm whether CRC patient is the same, theamount of Foxp3 was confirmed. The result showed that the regulatory Tcells were enriched in CRC patients (see Gut. 2011 Dec. 29, “Suppressionof tumour-specific CD4+ T cells by regulatory T cells is associated withprogression of human colorectal cancer”).

In addition, using Alloreactive regulatory T cells, the possibility ofconducting the organ transplant without continuous immunosuppressionwhich is the biggest problem of an organ transplant has been suggested(see Eur J Immunol 2011 March; 41 (3): 726-38 doi:10.1002/eji.201040509. Epub 2011 Jan. 17, “Induction of transplantationtolerance converts potential effector T cells into graft-protectiveregulatory T cells”).

The blood vessel where the regulatory T cells in cardiac fibrosis wastreated with adoptive transfer was significantly improved (see JHypertens 2011 September; 29 (9): 1820-8., see “CD4

CD25

Foxp3

regulatory T cells suppress cardiac fibrosis in the hypertensiveheart”.)

In order to confirm the influence of the regulatory T cells inAlzheimer's diseases, the regulatory T cell colonies of severe patients(Alzheimer Disease: AD) and mild patients were investigated. The resultshowed that the Treg expressing Program death receptor 1 was muchexpressed (see J Alzheimers Dis. 2010; 21(3):927-38, “PD1 negative andPD1 positive CD4+T regulatory cells in mild cognitive impairment andAlzheimer's disease”).

The regulatory T cells were activated using CD3 and then Parkinson'sDisease (PD) was treated using the Adoptive transfer of the regulatory Tcells (see J Leukoc Biol. 2007 November; 82 (5):1083-94. Epub 2007 Aug.3. “Neuroprotective activities of CD4+CD25+ regulatory T cells in ananimal model of Parkinson's disease”).

For vaccination promotion, especially for the vaccine developed usingpart or all of a variety of pathogens, when the vaccination is madethrough a specific route of administration (particularly for mucosalvaccine), the vaccine effect hardly appears in many cases. In this case,it has been found that since there existed many regulatory T cells inthe corresponding route of administration, the vaccine effect did notappear (see Journal of Immunology, 2007, 179, 5633-5638).

The compositions of the present invention may comprise, in addition tothe active ingredient, pharmaceutically suitable and physiologicallyacceptable adjuvants. These adjuvants include excipients, disintegratingagents, sweetening agents, binding agents, coating agents, swellingagent, lubricants, glidants and solubilizing agents.

In addition, the compositions of the present invention may be preferablyformulated and administered as a pharmaceutical composition comprisingat least one pharmaceutically acceptable carrier in addition to theabovementioned active ingredient. For the composition formulated as aliquid solution, a pharmaceutically acceptable carrier is suitable forsterilization and living body, which can be used by mixing saline,sterile water, Ringer's solution, buffered saline, albumin injectionsolution, dextrose solution, maltodextrin solution, glycerol, ethanoland one or more ingredients thereof. Also, the other conventionaladditives such as antioxidants, buffers and bacteriostatic agents may beadded as needed. Furthermore, the composition of the present inventionmay be formulated as injectable formulations, pills, capsules, granulesor tablets such as aqueous solution, suspension or emulsion by furtheradding diluents, dispersants, surfactants, binders and lubricants. Inaddition, the present composition may be preferably formulated,according to each disease or ingredient, using the methods disclosed inRemington's Pharmaceutical Science, Mack Publishing Company, Easton Pa.as a suitable method in the art.

In the composition of the present invention, the pharmaceuticalpreparation form may be granules, powders, coated tablets, tablets,capsules, suppositories, syrups, juices, suspensions, emulsions, dropsor injectable solutions and a sustained release preparation of theactive compound.

The compositions of the present invention may be administered by aconventional method via intravenous, intraarterial, intraperitoneal,intramuscular, intraarterial, intraperitoneal, intrasternal,percutaneous, intranasal, inhalation, topical, rectal, oral,intraocular, or intracutaneous routes.

In the treatment method of the present invention, the term “effectiveamount” refers to the amount required to achieve the effect ofsuppressing the cancer. Thus, the “effective amount” of the activeingredient of the present invention may be adjusted depending on variousfactors, including the type of diseases, the severity of diseases, thetype and content of the active ingredient and other components containedin the composition, the type of formulation, the age, body weight,general health condition, sex and diet of the patient, time ofadministration, route of administration and the secretion ratio of thecomposition, the period of treatment and the drug used simultaneously.For adults, the inhibitor of the gene or protein is preferablyadministered once to several times a day, at the following dosages: 0.01ng/kg˜10 mg/kg for siRNA, 0.01 ng/kg˜10 mg/kg for shRNA, 0.01 ng/kg˜10mg/kg for antisense oligonucleotides to mRNA of the gene, 0.1 ng/kg˜10mg/kg for the compound, 0.1 ng/kg˜10 mg/kg for the monoclonal antibodyto the protein, 0.1 ng/kg˜10 mg/kg for the small molecular compound, 0.1ng/kg˜10 mg/kg for the natural products, and 0.1 ng/kg˜10 mg/kg for thebioavailability protein.

Advantageous Effects

Lrig1 according to the present invention is specifically expressed onTreg, thereby controlling the Treg. Therefore, the present Lrig1 can beused as a new target of immunosuppressive agents.

DESCRIPTION OF DRAWINGS

FIG. 1 shows a comparison of the expression levels of Lrig1 innon-contacted T cells, Th1, Th2, Th17, iTreg and nTreg through Microarray.

FIG. 2 shows the difference of the expression level of the regulatory Tcell-specific Foxp3 gene as a positive control.

FIG. 3 shows a comparison of the expression level of Lrig1 innon-contacted T cells (CD44^(low), CD62L^(high)) and memory T cells(CD44^(high), CD62L^(low)) with the expression level of Foxp3+regulatory T cells through RT-PCRn (two times tests).

FIG. 4 shows a relative comparison of mRNA expression level of Lrig 1 inT cells.

FIG. 5 (a) confirms the expression of Lrig 1 in CD4⁺CD25^(high) cells,CD4⁺ CD25^(low) cells and CD4⁺Foxp3⁺ cells through Western blot, and (b)when CD4⁺CD25^(high) cells and CD4⁺CD25^(low) cells were compartmentedinto only CD4 positive cells, compares these cells with the controlantibody in each cell group, analyses the expression level of LRIG1 withFACS and shows the difference (%). It can be seen that the expression ofLrig1 is increased in the natural regulatory T cells (nTreg) (from 0% to6.24%).

FIG. 6 (a) confirms the expression reduction of LRIG1 by the reversetranscription polymerase chain reaction after delivering control siRNAand Lrig1-specific siRNA to proliferated CD4⁺Foxp3⁺, and (b) it can beseen that the regulatory T cells with reduced expression of LRIG1 doesnot greatly increase its number upon expansion. Here, the blue showsthat expansion (proliferation) of Treg cells is not increased when Tregcells (CD4⁺CD25^(high)) were treated with Lrig1-specific siRNA and thatthe non-treated Treg cells (red) were normally expanded. The blue isindicated as (siRNA+CD4⁺CD25^(high)) and the red is indicated as(CD4⁺CD25^(high)).

FIG. 7 shows that, when naive T cells (CD⁺CD25¹′) stimulated withanti-CD3+anti-CD28mAb was cultured as in Treg cells (CD4⁺CD25^(high)),the proliferation of activated T cells was effectively inhibited in Tregcells with high Lrig-1 expression in mixed lymphocyte reaction, but Tregcells with lowered Lrig-1 expression by treatment of Lrig 1-specificsiRNA did not inhibit the expansion of cells. As such, this shows therelationship between the expression and immunosuppressive activity ofLRIG1 in CD4⁺CD25^(high) cells (A). By re-confirming the study result of(A) using CFSE dilution reaction method, the expression LRIG1 inCD4⁺CD25^(hi)g cells is proven to be important in the immunosuppressiveactivity of Treg cells.

FIG. 8 shows RT-PCR test result using LRIG1-specific primer wherein Bcells, CD8+T cells, naive CD4+T cells, effector CD4+T cells and Tregcells were isolated in mouse to isolate mRNA and then they are used as atemplate. It can be seen from the test result that LRIG1 is specificallymuch expressed in the regulatory T cells.

FIG. 9 shows RT-PCR test result using LRIG1-specific primer whereinnaive CD4+T cells, effector CD4+T cells and Treg cells were isolated inmouse to isolate mRNA and then an activation signal is given to thecells by anti-CD3+anti-CD28 mAb stimulation, mRNA is separated from eachcell and they are used as a template. It can be seed from the testresult that LRIG1 is specifically much expressed in the regulatory Tcells and that the expression in the activated regulatory T cells ishighly induced.

FIG. 10 and FIG. 11 are about the result wherein the proteins of severalstimulated cells are subjected to Western blotting with mAb to LRIG1d,and the cell colonies through their FACS.

MODE FOR INVENTION

Advantages and features of the present invention and methods ofaccomplishing the same will be apparent with reference to the examplesset forth in detail below. However, the present invention is notintended to be limited to the examples set forth herein, and it may beimplemented in many different forms. These examples are only provided tocomplete the disclosure of the present invention and to sufficientlydefine the scope of the invention to a person with ordinary skill in thetechnical field to which the present invention pertains. The presentinvention will be only defined by the appended claims.

Example 1

Isolation and Culture of T Cells

C57BL6 (H-2^(b)) wild-type mice and Foxp3-GFP transgenic mice were used.All mice were bred in a clean condition (SPF) where the temperature andhumidity were properly adjusted, and was used at the period of 8-12 weekage in accordance with the animal experiment protocol approved by theAnimal Experiment Commission of Yonsei University in Seoul.

The spleen cells of C57BL/6 rats were isolated to which the antibodiesattached with anti-CD4-magnetic bead was added and passed through amagnetic activated cell analyzer (MACS) (MiltenyiBiotec), thus isolatingCD4+ cells (90-95%). Nest, in order to conduct MicroArray, the CD4+cells were stained with anti-CD4-antibody (Clone RM4-5, BD bioscience),anti-CD25− antibody (Clone 7D4, BD bioscience), anti-CD44− antibody(Clone IM7, BD bioscience) and anti-CD62L-antibody (Clone MEL-14, BDbioscience), and then non-contacted T cells (Naive T cells),CD4⁺CD62L⁺CD44⁻CD25⁻, natural regulatory T cells (nTreg),CD4⁺CD25^(high) cells and CD4⁺CD25^(low) cells were isolated through theflow cytometry (FACS, BD FACSAria™II).

The T cells were removed through non-contacted T cells, anti-Thy1.2antibody and rabbit complement (Cedarlance Laboratories Limited) towhich Antigen-Presenting Cells (APCs) isolated by radiation of 30Gy(3000 rad) were added at a ratio of 1:5 and stimulated with anti-CD3antibody 1 μg/

(clone 145-2C11, BD bioscience) and anti-CD28 antibody 3 μg/

(clone 37.51, BD bioscience). Next, the antibody differentiating intoeach group of the T cells was added with cytokines and the cells werecultured for 72 hours. IL-2 (100 U/ml was added to the remaining cellgroup except for iTreg and further cultured for 4 days. The type andamount of antibodies for each group of cells were as follows:

Th1-anti-IL-4 (10 μg/

), IL-12 (10 ng/

), IL-2 (100 U/

)

Th2-anti-IFN-g (10 μg/

), anti-IL-12 (10 ng/

), IL-4 (5000 U/

) and IL-2 (100 U/

)

Th17-anti-IL-4 (10 μg/

), anti-IFN-410 μg/

), anti-IL-12 (10 μg/

), TGF-β(5 ng/

), IL-6 (10 ng/

), IL-1 (3(10 ng/

) iTreg-anbti-IL-4 (10 μg/

), anti-IFN-410 μg/

), anti-IL-12 (10 μg/

), TGF-β(5 ng/

), IL-2 (100 U/

)

In order to conduct a reverse transcription polymerase chain reaction(RT-PCR, Reverse transcription-PCR), the total RNA was extracted usingkit (Allprep DNA/RNA Mini Kit™ Qiagen) in CD4⁺CD25^(low) cells andCD4⁺CD25^(hi)g cells and then the complementary DNA (cDNAs) weresynthesized using kit (Transcriptor High Fidelity cDNA Synthesis Kit™Roche). The polymerase chain reaction was carried out usingoligonucleotide primers and PCR composition (master mix, Qiagen) wherethe purified complementary DNBA was directly designed. The transcriptionfactor foxp3 and gamma actin 1 (ACTG1) of the regulatory T cells (Treg)were used as a control group. Each primer sequence is as follows:

TABLE 1 Primers for protein amplification Protein SEQ ID NO SequenceDirection LRIG1 1 ACCACCGTAGGCATCTTCAC Forward 2 GAGCCACTGTGTGCTGTTGTReverse FOXP3 3 CCCTTGGCCCATCCCCAGGA Forward 4 CCGAGCGTGGGAAGGTGCAGReverse ACTG1 5 GGCGTCATGGTGGGCATGGG Forward 6 ATGGCGTGGGGAAGGGCGTAReverse

Example 2

Identification of Lrig-1 Surface Protein Specifically Expressing toCD4⁺CD25⁺ nTreg

Cells isolated from Example 1, i.e., CD4⁺CD25^(high) cells, CD4⁺CD25^(low) cells and CD4⁺Foxp3⁺ cells were dissolved with RIPA cytolysisbuffer (Sigma). The same amount of cell suspension was then subjected toelectrophoresis through SDS PAGE gel and moved into the membrane.Western blot was conducted using anti-LRIG1-antibody (SantaCruz),anti-Foxp3-antibody (ebioscience), and anti-beta actin (Cell Signaling).

To the cells isolated with a flow cytometry through a surface proteinstaining, i.e., CD4⁺CD25^(high) cells and CD4⁺CD25^(low) cells,anti-LRTG-antibody (R&D systems) and control antibody (Goat-IgG control,R&D systems) were added to compare the expression level of LRIG, reactedat a temperature of 4r for 30 minutes and then washed with FACS buffer(0.05% sodium azide, 0.5% BSA/PBS). The flow cytometric analysis wasconducted with the flow cytometry (FACSCalibur, BD Bioscience).

Example 3

Identification of the Relationship Between Lrig-1 Expression in Treg andTreg Proliferation

Mouse Lrig1-specific siRNAs (Thermo Fisher Scientific) was prepared intoa liposome-siRNA complex (Lipofectamin 2000™ Invitrogen) and the amountof final siRNA was made as 5-50 pmol and then transfected inproliferated CD4⁺Foxp3⁺ T cells 1×10⁵/2

cell culture solution. After 24 hours, the reduction of expression wasconfirmed through the reverse transcription polymerase chain reaction.The Lrig1-specific siRNAs sequences used are as follows:

TABLE 2 siRNA for expression inhibition SEQ ID NO Name Sequence 7SMARTpool siRNA CCGAACGGCCUGCGUAUAA J-046693-09 8 SMARTpool siRNAGGAGCCAGCUGAAGUCGUA J-046693-10 9 SMARTpool siRNA GGUCUGUAGUUGAGGACGAJ-046693-11 10 SMARTpool siRNA CCUGGAAGGUGACGGAGAA J-046693-12

Example 4

Identification of Relationship Between the Expression Reduction ofLrig-1 in Treg and the Suppressive Activity of Treg

<Method for Suppressing a Mixed Lymphocyte Culture Reaction>

2.5×10⁵ of C57BL/6 mouse-derived CD4⁺CD25^(low) T cells per each wellwere introduced in the 96-well plate immobilized with anti-CD3 antibody(clone 145-2C11) (5 μg/

) to which anti-CD28 antibody (clone 37.51) (2.5 μg/

) in an aqueous solution state was added and stimulated. The stepwisediluted nTreg and siRNA were added thereto. After one day, nTreg wasadded and subjected to mixed reaction in 5% CO₂ incubator at 37° C. for72 hours. Before the final 6 hours, 1 μci of [³H]-thymidine (185GBq/mmol, Amersham Biosciences) per well was added. The cells werecollected on filter paper using a cell harvester. The amount of[³H]-thymidine incorporated within the cells was measured by TopCountNXT beta counter (PerkinElmer). Hence, it can be seen that theexpression of LRIG1 in CD4⁺CD25^(high) cells is important in theimmunosuppressive activity through the incorporation of [³H]-thymidineafter a mixed lymphocyte culture reaction (see FIG. 7 (a)).

<CFSE Dilution Reaction (Carboxyfluorescein Succinimidylester)>

The CD4⁺CD25^(low) cells were pre-labeled with 104 CFSE (Sigma), andthen 1×10⁶ cells per well were introduced in 96-well plates immobilizedwith anti-CD3 antibody (clone 145-2C11) (5 μg/

) and then stimulated in a CO₂ incubator at 37° C. for 48 or 96 hours.Also, anti-CD28-antibody (clone 37.51) (2.5 μg/

) in an aqueous solution state was added and stimulated. The stepwisediluted nTreg and siRNA were added thereto. After one day, nTreg wasadded and subjected to mixed reaction in 5% CO₂ incubator at 37° C. for72 hours. The anti-CD3 antibody (clone 4-5, BD bioscience) was added,reacted at a temperature of 4° C. for 30 minutes, washed with FACSbuffer (0.05% sodium azide, 0.5% BSA/PBS). Before 10 minutes of theanalysis, 7-AAD (ebioscience) was added, thus analyzing only the livingcell. The CFSE dilution in CD4⁺ cells was measured through FACSCalibur(BD Bioscience) to investigate the cell frequency and the proliferationtimes where the cell proliferation occurs. As a result, CD4⁺CD25^(low)cells stained with CFSE were subjected to mixed lymphocyte reaction. Itcan be seen that the expression of LRIG1 in CD4⁺CD25^(high) cells isimportant in the immunosuppressive activity through the CFSE dilutiondegree (see FIG. 7 (b)).

Example 5

Identification of Human Treg Cell-Specific Expression

Cells stimulated with anti-CD3+anti-CD28 mAb by isolation of human naiveT cells, cells differentiated with iTreg by the treatment ofanti-CD3+anti-CD28 mAb+TGFb+IL-2, cells treated with Rapamycin orEverolimus (inhibitors to inhibit proliferation of cells induced proteinfunctions of mTOR and the derivatives thereof) known to induce theproliferation of Treg with the treatment of anti-CD3+anti-CD28mAb+TGFb+IL-2, Jurkat human T cells, and naive human CD4+T cells werestimulated with anti-CD3+anti-CD28 mAb to isolate activated cells. Theprotein was prepared from each of these cells. Western blotting wasconducted with mAb to LRIG1d. As a result, it can be seen that LRIG1dwas specifically expressed only on iTreg and that the expression levelis markedly increased in the activation and proliferation of Treg andproliferated iTreg. It has been shown that LRIG1 was greatly expressedeven in the regulatory T cells of rats as well as in the regulatory Tcells of human, particularly, the expression was increased in theactivated or proliferated regulatory T cells (see FIG. 10 and FIG. 11).

1. A method for treating immune-related diseases comprisingadministering an effective amount of a regulatory T cell to a subjectwith immune-related diseases, wherein the regulatory T cell has Lrig-1overexpression.
 2. The method of claim 1, the immune-related diseasesare one selected from the group consisting of autoimmune diseases, graftversus host diseases, organ transplant rejection, asthma, atopicdermatitis, acute and chronic inflammatory diseases, cardiovasculardiseases and cognitive disorders.
 3. The method of claim 1, theproliferation of activated T cells is inhibited by the Treg cellsoverexpressing Lrig-1 protein.